This proposal focuses on the molecular bases of two inherited diseases in man, Beta+ thalassemia and orotic aciduria. Homozygous Beta+ thalassemia is usually a severe anemia characterized by reduced levels of beta-globin mRNA and protein synthesis. The thalassemias of T.T. and several other patients have been shown to be due to inefficient processing of the Beta-globin mRNA precursor to mRNA. The removal of one or both intervening sequences present in the primary transcripts of these patients' Beta-globin alleles takes place incorrectly and/or abnormally slowly so that Beta-globin RNA processing intermediates accumulate at the expense of mRNA production. T.T. is unusual in that she is only mildly anemic. The correlation between the precise lesion within a thalassemic Beta-globin allele and the efficiency of functional Beta-globin mRNA production is not known. To investigate this correlation, I propose to (1) use recombinant DNA technology and DNA sequencing to localize the lesion(s) within the Beta-globin alleles of T.T., and (2) obtain expression of T.T.'s cloned Beta-globin alleles in HeLa cells for purposes of structurally characterizing the Beta-globin RNA processing intermediates that accumulate. Techniques will include RNA blotting and S1-nuclease transcript mapping. Orotic aciduria results from a deficiency in two enzymes of pyrimidine nucleotide biosynthesis, orotate phosphoribosyltransferase (OPRT) and orotidine-5 feet-phosphate decarboxylase (ODC). The disease is characterized by infantile megaloblastic anemia and urinary orotic acid accumulation. Data support the existence of a functional genetic element shared by the genes encoding OPRT and ODC. With the goal of cloning and characterizing the DNA sequence(s) and cellular process(es) altered in orotic aciduria, I propose to (1) isolate human DNA that confers uridine-independent growth to a Urd-C Chinese hamster ovary cell line (that is deficient in OPRT and ODC and, thus, is dependent on exogenous uridine for growth), (2) examine the transcription, RNA processing, and translation of complementary sequences in fibroblasts cell lines of normal individuals, and (3) purify, characterize and examine the expression of analogous DNA sequences in cultured fibroblasts of patients with orotic aciduria. This work should contribute to our understanding of normal gene expression and the molecular mechanisms responsible for certain aberrant phenotypes in man.